We have identified a novel enzyme designated PAS-kinase (PASK). The human version of PASK is encoded by a single gene located on chromosome 2, band q37. Analysis of genomic and cDNA clones of PASK has shown the gene to be composed of 18 coding exons covering roughly 37 kilobases of human chromosome 2. The enzyme is 1,323 amino acid residues in length and highly related in primary sequence to polypeptides specified in the genomes of flies and yeast. RT-PCR assays conducted on multiple tissues of the mouse indicate that PASK mRNA is expressed at a similar level in all tissues.
As shown in Table 1 (below), the polypeptide sequences of the human, fly and yeast versions of PASK are most conserved in three regions. Two of the three conserved regions correspond to PAS domains, designated PAS-A and PAS-B, the former being located closer to the amino terminus of the enzyme. The third conserved region of PASK encodes a serine/threonine kinase domain. When compared with all entries available in the database of protein kinases kept by Tony Hunter [1], PASK is most similar in primary amino acid sequence to the catalytic domains of the 5' AMP activated protein kinase (AMPK), its yeast ortholog SNF1, and the product of the Pim-1 oncogene.
We conducted a two-pronged approach to resolve the biological function of PASK. Biochemical experiments were carried out to study the catalytic activity of the enzyme as well as the modulatory role enacted by the two PAS domains. Complementary genetic experiments were undertaken to study the biological role of PASK in budding yeast. The latter experiments have also directed preliminary studies pertinent to the role of PASK in mammalian cells. In summary, these efforts have established PASK as a serine/threonine kinase that is regulated in cis by its two PAS domains. They have likewise implicated PASK in the regulation of translation and the balance of cell growth (cell size) and mitosis. Biophysical studies of the two PAS domains of PASK have identified PAS-A as a ligand-binding regulatory domain of the PASK enzyme. Together these studies reveal an entirely novel and unanticipated regulatory system that represents a valuable target for the development of synthetic organic compounds capable of regulating the mitotic growth of mammalian cells.
Limited aspects of this disclosure were presented at the 19th International Conference on Magnetic Resonance in Biological Systems at Florence, Italy in August 2000; see, Structural studies of PAS domains: Insight into the link between ligand and protein binding, K H Gardner, C A Amezcua and S M Harper, Aug. 25, 2000, 19.sup.th Intrnl Conf Magnetic Resonance in Biological Systems, Florence, Italy; and Solution structure and dynamics of a eukaryotic PAS domain: Evidence for a flexible ligand binding region, C A Amezcua, S M Harper and K H Gardner, Aug. 21, 2000, 19.sup.th Intrnl Conf Magnetic Resonance in Biological Systems, Florence, Italy. Genbank accession KIAA0135 has sequence similarity to the disclosed human PASK.